The present invention relates to the field of modulating cell apoptosis, particularly agents useful to inhibit apoptosis, as well as to diagnostic and prognostic assays involving conditions in mediated by the expression of inhibitors of apoptosis. The invention specifically relates to the identification of a novel human gene, tentatively named Survivin. Survivin encodes a protein, Survivin, that inhibits cellular apoptosis, particularly in cancer cells and embryonic cells.
Regulation of cell proliferation by programmed cell death (apoptosis) maintains tissue homeostasis during development and differentiation (Raff, M. D., Nature (1992) 356:397400; Vaux, D. L. et al., Cell (1994) 76:777-779). This process involves an evolutionarily conserved multi-step cascade (Oltvai, Z. et al., Cell (1994) 79:189-192), and is controlled by proteins that promote or counteract apoptotic cell death Apoptosis also involves cell surface receptors (Smith, A. et al., Cell (1994) 76, 959-962), and associated signal transducers (Tartaglia, L. A. et al., Immunol Today (1992) 13:151-153), protease gene families (Martin, S. J. et al., Cell (1995) 82:349-352), intracellular second messengers (Kroemer, G. et al., FASEB J (1995) 9:1277-1287), tumor suppressor genes (Haffner, R. et al., Curr Op Gen Dev (1995) 5:84-90), and negative regulatory proteins that counteract apoptotic cell death (Hockenbery, D. et al., Nature (1990) 348:334-336). Aberrantly increased apoptosis or abnormally prolonged cell survival (Oltvai, Z. N. et al., Cell (1994) 79:189-192) may both contribute to the pathogenesis of human diseases, including autoimmune disorders, neurodegenerative processes, and cancer (Steller, H., Science (1995) 267:1445-1449; Thompson, C. B., Science (1995) 267:1456-1462).
Specifically, for example, inhibitors of apoptosis, most notably of the bcl-2 family (Reed, J, J Cell Biol (1994) 124:1-6, and Yang, E, et al., Blood (1996) 88:386-401), maintain lymphoid homeostasis and morphogenesis in adult (Hockenbery, D et al., Proc Natl Acad Sci USA (1991) 88:6961-6965) and fetal (LeBrun, D. et al. (1993) 142:743-753) tissues. Deregulated expression of bcl-2 has also been implicated in cancer, by aberrantly prolonging cell survival and facilitating the insurgence of transforming mutations.
In addition to bcl-2, several members of a new gene family of inhibitors of apoptosis related to the baculovirus IAP gene (Bimbaum, M. J. et al., J Virology (1994) 68:2521-2528; Clem, R. J. et al., Mol Cell Biol (1994) 14:5212-5222) have been identified in Drosophila and mammalian cells (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A. et al., Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al., Cell (1995) 80:167-178). These molecules are highly conserved evolutionarily; they share a similar architecture organized in two or three approximately 70 amino acid amino terminus Cys/His baculovirus IAP repeats (BIR) and by a carboxy terminus zinc-binding domain, designated RING finger (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A. et al., Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al., Cell (1995) 80:167-178). Recombinant expression of IAP proteins blocks apoptosis induced by various stimuli in vitro (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Liston, P. et al., Nature (1996) 379:349-353), and promotes abnormally prolonged cell survival in the developmentally-regulated model of the Drosophila eye, in vivo (Hay, B. A. et al., Cell (1995) 83:1253-1262). Finally, deletions in a IAP neuronal inhibitor of apoptosis, NAIP, were reported in 75% of patients with spinal muscular atrophy, thus suggesting a potential role of this gene family in human diseases (Roy, N. et al., Cell (1995) 80:167-178).
Therapeutic and diagnostic uses of nucleic acids that encode various inhibitors of apoptosis relating to a member of the IAP family have been described in the patent literature. See, for example, International Patent Applications No. WO 97/06255, WO 97/26331, and WO 97/32601. In particular, the uses of such genes and gene products are contemplated for the novel protein and its encoding nucleic acid discussed below.
Recently, a novel gene encoding a structurally unique IAP apoptosis inhibitor, designated Survivin has been identified. Survivin is a xe2x88x9216.5 kD cytoplasmic protein containing a single BIR, and a highly charged carboxyl-terminus coiled-coil region instead of a RING finger, which inhibits apoptosis induced by growth factor (IL-3) withdrawal when transferred in B cell precursors (Ambrosini, G. et al., Nature Med. (1997) 3:917-921). At variance with bcl-2 or other IAP proteins, Survivin is undetectable in adult tissues, but becomes prominently expressed in all the most common human cancers of lung, colon, breast, pancreas, and prostate, and in xe2x88x9250% of high-grade non-Hodgkin""s lymphomas, in vivo. Intriguingly, the coding strand of the Survivin gene was highly homologous to the sequence of Effector cell Protease Receptor-1 (EPR-1) (Altieri, D. C., FASEB J (1995) 9:860-865), but oriented in the opposite direction, thus suggesting the existence of two separate genes duplicated in a head-to-head configuration.
The present invention is based on the identification of a novel human gene which is nearly identical to EPR-1, but oriented in the opposite direction. The antisense EPR-1 gene product, designated Survivin, is a distantly related member of the IAP family of inhibitors of apoptosis (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A. et al., Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al., Cell (1995) 80:167-178), and is prominently expressed in actively proliferating transformed cells and in common human cancers, in vivo, but not in adjacent normal cells. Functionally, inhibition of Survivin expression by up-regulating its natural antisense EPR-1 transcript resulted in massive apoptosis and decreased cell growth.
The present invention is based, in part, on the isolation and identification of a protein that is expressed in most cancer cells and inhibits cellular apoptosis, hereinafter Survivin or the Survivin protein. Based on this observation, the present invention provides purified Survivin protein.
The present invention further provides nucleic acid molecules that encode the Survivin protein. Such nucleic acid molecules can be in an isolated form, or can be operably linked to expression control elements or vector sequences.
The present invention further provides methods of identifying other members of the Survivin family of proteins. Specifically, the nucleic acid sequence of Survivin can be used as a probe, or to generate PCR primers, in methods to identify nucleic acid molecules that encode other members of the Survivin family of proteins.
The present invention further provides antibodies that bind to Survivin. Such antibodies can be either polyclonal or monoclonal. Anti-Survivin antibodies can be used in a variety of diagnostic formats and for a variety of therapeutic methods.
The present invention further provides methods for isolating Survivin binding partners. Survivin binding partners are isolated using the Survivin protein as a capture probe. Alternatively, Survivin can be used as bait in the yeast two-hybrid system to screen an expression library and identify genes that encode proteins that bind to the Survivin protein Binding partners isolated by these methods are useful in preparing antibodies and also serve as targets for drug development.
The present invention further provides methods to identify agents that can block or modulate the association of Survivin with a binding partner. Specifically, an agent can be tested for the ability to block, reduce or otherwise modulate the association of Survivin with a binding partner by contacting Survivin, or a fragment thereof, and a binding partner with a test agent and determining whether the test agent blocks or reduces the binding of the Survivin protein to the binding partner.
The present invention further provides methods for reducing or blocking the association of Survivin with one or more of its binding partners. Specifically, the association of Survivin with a binding partner can be blocked or reduced by contacting Survivin, or the binding partner, with an agent that blocks the binding of Survivin to the binding partner. The method can utilize an agent that binds to Survivin or to the binding partner.
The present invention further provides methods of regulating the expression of Survivin within a cell. Expression of Survivin within a cell can be regulated so as to produce or inhibit the production of Survivin.
Blocking Survivin/binding partner associations or Survivin expression can be used to modulate biological and pathological processes that require Survivin. For example, methods that reduce Survivin production induce apoptosis of tumor cells. Stimulation of Survivin production can be used as a means of extending the culturability of cells or tissues.
The biological and pathological processes that require Survivin or Survivin/binding partner interactions can further be modulated using gene therapy methods. Additional genetic manipulation within an organism can be used to alter the expression of a Survivin gene or the production of a Survivin protein in an animal model. For example, a Survivin gene can be altered to correct a genetic deficiency; peptide modulators of Survivin activity can be produced within a target cell using genetic transformation methods to introduce a modulator encoding nucleic acid molecules into a target cell; etc. The use of nucleic acids for antisense and triple helix therapies and interventions are expressly contemplated.
The present invention further provides methods of reducing the severity of pathological processes that require Survivin. Since expression of Survivin or association of Survivin with a binding partner is required for Survivin-mediated biological processes, agents that block Survivin expression, Survivin activity or the association of Survivin with a binding partner, can be used in therapeutic methods.